TIROSIN CHINASI CITOPLASMATICHE

SH3 SH2 PH SH2 SH3 SH2 SH2 SH3 SH2 TIROSIN CHINASI CITOPLASMATICHE KINASE DOMAIN PH Tec (Btk/Itk/Bmx) KINASE DOMAIN FAK/Pyk2 Focal adhesion targeting (paxillin binding) (FERM) KINASE DOMAIN SH2 Jak Src (Blk, Lck, Lyn, Fyn, Fgr, Hck, Yes) KINASE DOMAIN SH3 SH2 Ac. miristico/palmitico KINASE DOMAIN SH2 ZAP70/Syk Abl/Arg KINASE DOMAIN SH3 SH2 Ac. miristico

G. Steven Martin : THE HUNTING OF THE SRC, Nature Reviews Molecular Cell Biology 2, 467-475 (2001)  

Integrine Src

di molecole adesive definite INTEGRINE FAK e membri della famiglia del Src costituiscono un’unità di trasduzione del segnale da parte di molecole adesive definite INTEGRINE SH3 SH3 SH2 YP397 Src FAK Src SH2 SH1 SH1 FAK

Outside-in signaling

FAK F1 F2 F3 SH2 SH2 N C SH3 YP Autofosforilazione Interazioni con altre proteine (Src, PI3 chinasi, PLCg) SH2 YP Sito di interazione con Grb2 SH2 F1 F2 F3 C dominio catalitico dominio FERM dominio FAT N Poli-P Interazione con Cas SH3 Interazione con catena b delle integrine e fattori di crescita Interazione con fosfatidil inositolo (4,5) bisfosfato (PIP2) Interazione con proteine favorenti la polimerizzazione dell’actina (complesso Arp 2/3) Interazione con paxillina

la mantengono in uno stato inibito. Anche FAK come Src viene attivata in seguito alla rottura di interazioni intermolecolari che la mantengono in uno stato inibito.

Il distacco da proteine della matrice extracellulare induce una forma di apoptosi (chiamata “anoikis”) che determina il fenomeno della cosiddetta “crescita dipendente dall’ancoraggio” SH3 SH2 YP397 Src FAK AKT PI3K PY PROLIFERAZIONE E SOPRAVVIVENZA

FAK Cas Fosfatidilinositolo 4,5 fosfato P P P P SH3 P P P P P P PH SH2 PI3-chinasi YP397 Src IP3 PIP5-chinasi g FAK PLCg GEFs SH1 PY Cas paxillina PY PY YP925 Rac, Cdc42 Poli-P Crk Poli-P Dock180 Grb2 Sos Rac Interazione con altre Proteine cito- scheletriche e segna- lanti Ras PAK Polimerizzazione dell ’actina e protrusione del lamellopodio JNK Raf MAPK MAPK (ERK1/2) Movimento cellulare Proliferazione e sopravvivenza cellulare TRASCRIZIONE GENICA

FAK, Src e altre chinasi vengono inibite da tirosin fosfatasi che sono sensibili all’azione inibitoria di ROIs FAK, Src, GF-Rs, MAP chinasi (JNK, ERKs) attive FAK, Src, GF-Rs, MAP chinasi (JNK, ERKs) inattive Protein fosfatasi inattiva Protein fosfatasi attiva

La transizione epitelio-mesenchimale rappresenta una modificazione tipica dei tumori epiteliali metastatici An epithelial-mesenchymal transition (EMT) is a biologic process that allows a polarized epithelial cell, which normally interacts with basement membrane via its basal surface, to undergo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype, which includes enhanced migratory capacity, invasiveness, elevated resistance to apoptosis, and greatly increased production of ECM components . The completion of an EMT is signaled by the degradation of underlying basement membrane and the formation of a mesenchymal cell that can migrate away from the epithelial layer in which it originated. (Kalluri and Weinberg, J. Clin. Invest. 119:1420, 2009)

EPITHELIAL-MESENCHIMAL TRANSITION MESENCHIMAL-EPITHELIAL TRANSITION

E-caderina E-caderina giunzioni serrate (tight junction) F-actina microtubuli p120 cat. Previene endocitosi e de- gradazione E- caderina giunzioni aderenti (adherens junctions) E-caderina p120 cat. E-caderina b cat. vinculina Rac a cat. F-actina Rho b cat. Movimento Migrazione nel nucleo e trascrizione geni implicati nella proliferazione cellulare

Recettori per fattori di crescita: TGFbRI e II, IGF-1R, EGFR, c-Met L’aumentata stimolazione con fattori di crescita altera funzioni di E-caderine In diversi modi Recettori per fattori di crescita: TGFbRI e II, IGF-1R, EGFR, c-Met giunzioni serrate (tight junction) F-actina giunzioni aderenti (adherens junctions) p120 cat. E-caderina b cat. vinculina a cat. F-actina

L’aumentata stimolazione con fattori di crescita altera funzioni di E-caderine In diversi modi Il promotore del gene per le E-caderine viene represso da repressori trascrizionali (Snail, Twist, Slug e altri) indotti da Recettori per GF e altre vie attivate nel corso di “progressione” tumorale (compreso Nf-kB). 2. Tirosin chinasi recettoriali (EGFR, IGF-1R, c-Met, FGF) e non recettoriali (c-Src) inducono fosforilazione e successiva ubiquitinilazione di E-caderine e catenina con conseguente degradazione nel proteosoma. La fosforilazione di E-caderina comporta anche il distacco di a e b catenine. 3. Proteasi (metallo-proteasi – MMP) proteolizzano E-caderine alterando contatti cellula-cellula. 4. Gli stessi segnali che “down-regolano” espressione di E-caderine aumentano espressione di N-caderine (il cosiddetto “caderin-switch”), che sono espresse da cellule mesenchimali e regolano positivamente il movimento cellulare.

movimento Sress fibers, focal adhesion Cdc42 Rac Rho p120 cat. vinculina E-caderina a cat. F-actina b cat.

Inibizione di GSK da parte di GF-Rs o altri stimoli favorisce la progressione neoplastica Snail P GSK-3 b cat. E-caderina b cat. P Repressione sintesi E-caderine Trascrizione geni implicati in proliferazione e progressione DEGRADAZIONE NUCLEO

Un meccanismo aggiuntivo di regolazione della funzione di bcatenica è rappresentato da un suo aumento di espressione in cellule stimolate con proteine wnt (ampia famiglia di fattori di crescita poco caratterizzati) Gene onco-soppressore, la cui mutazione in cellule della linea germinale determina la Poliposi Adenomatosa Famigliare del Colon

a | In the absence of WNT signalling, -catenin levels in the cytoplasm and nucleus are low as a result of continuous phosphorylation by the serine/threonine kinases CK1 (casein kinase 1) and GSK3 (glycogen synthase kinase 3), leading to binding of -transducin-repeat-containing protein (TRCP) and to ubiquitylation and degradation by the proteasome. The destruction complex is composed of CK1 and GSK3, as well as the anchor proteins AXIN1 (axis inhibition protein 1 ) and APC (adenomatous polyposis coli). In the nucleus, TCF (T-cell factor) molecules are bound by co-repressors such as GRG/TLE (Groucho/transducin-like enhancer) proteins that shut off expression of WNT target genes. Other components of the repressor complex include CTbP (C-terminal binding protein) and HDACs (histone deacetylases). -catenin in the nucleus is inhibited from binding TCF by ICAT (cell autonomous inhibitor of -catenin and TCF) . The Frizzled receptor complex — composed of Frizzled and LRP5 (LDL-receptor-related protein 5) or LRP6 — can also be actively inhibited by receptor-bound soluble inhibitors such as DKK1 (Dickkopf homologue 1). b | Upon binding of a lipid-modified WNT protein to the receptor complex, a signalling cascade is initiated. LRP is phosphorylated by CK1 and GSK3, and AXIN1 is recruited to the plasma membrane. The kinases in the -catenin destruction complex are inactivated and -catenin translocates to the nucleus to form an active transcription factor complex with TCF, leading to transcription of a large set of target genes. In the nucleus, -catenin binds to TCF and LEF factors and recruits co-factors such as legless (LGS; also known as BCL9) and Pygopus (PYGO), CBP/p300, Brahma and MED12 to initiate transcription99, 100, 101. DVL, mammalian homologue of Drosophila Dishevelled; PP2A, protein phosphatase 2A.

WNT PROTEINS POSSONO ANCHE ATTIVARE RHO GTPASI E SEGNALI CALCIO

a | Planar cell polarity (PCP) signalling does not involve -catenin, LRP (LDL-receptor-related protein) or TCF (T-cell factor) molecules, but leads to the activation of the small GTPases RHOA (RAS homologue gene-family member A) and RAC1, which activate the stress kinase JNK (Jun N-terminal kinase) and ROCK (RHO-associated coiled-coil-containing protein kinase 1) and leads to remodelling of the cytoskeleton and changes in cell adhesion and motility. Through largely unknown mechanisms, canonical -catenin signalling can be inhibited by the PCP pathway. b | WNT–Ca2+ signalling is mediated through G proteins and phospholipases and leads to transient increases in cytoplasmic free calcium that subsequently activate the kinases PKC (protein kinase C) and CAMKII (calcium calmodulin mediated kinase II) and the phosphatase calcineurin. The activation of PLC (phospholipase C) by DVL (mammalian homologue of Drosophila Dishevelled) leads to the cleavage of PtdIns(4,5)P2 (phosphatidylinositol-4,5-bisphosphate) into InsP3 (inositol trisphosphate) and DAG (diacylglycerol). DAG, together with calcium, activates PKC, whereas InsP3 binding to receptors on the membranes of intracellular calcium stores leads to a transient increase in cytoplasmic free calcium, often also triggering an increase from extracellular stores. AP1, activator protein 1; CDC42, cell-division cycle 42; DAAM, Dishevelled-associated activator of morphogenesis; NFAT, nuclear factor of activated T cells; PDE6, phosphodiesterase 6.

Dipartimento di Patologia Sezione di Patologia Generale Università di Verona

Chronic myeloid leukemia-blast phase Granulocyte/macrophage progenitors Megakaryocyte/erythrocyte progenitors Common myeloid progenitors Haematopoietic stem cell Common lymphoid progenitors Chronic myeloid leukemia-blast phase (70%) Chronic myeloid leukemia-blast phase (30%) Chronic myeloid leukemia-chronic phase R. Ren, 5:172-183

Weisberg et al. 7:345, 2007 With the aid of several mediator proteins, BCR-ABL associates with Ras and stimulates its activation. The adaptor protein, growth factor receptor-bound protein 2 (GRB2), interacts with BCR-ABL through the proximal SRC homology 2 (SH2)-binding site that develops when the tyrosine 177 (Y177) residue of BCR-ABL is autophosphorylated. GRB2, when bound to BCR-ABL, interacts with the son of sevenless (SOS) protein. The resulting BCR–ABL–GRB2–SOS protein complex activates Ras. The adaptor proteins CRKL (CRK-like) and SHC (SH2-containing protein) can also mediate the BCR-ABL activation of Ras. Ras and the mitogen activated protein kinase (MAPK) pathway are coupled by Raf (a serine/threonine kinase). Raf catalyses the phosphorylation of the mitogen-activated and extracellular-signal regulated kinase kinases 1 and 2 (MEK1 and MEK2); this results in their activation. Through the stimulation of the Ras–Raf pathway, BCR-ABL increases growth factor-independent cell growth. BCR-ABL also associates with and activates the phosphatidylinositol-3 kinase (PI3K) pathway, suppressing programmed cell death and increasing cell survival. BCR-ABL is associated with components of the focal adhesion(that is, actin, paxillin and focal adhesion kinase, or FAK); the activation of CRKL–FAK–PYK2 leads to a decrease in cell adhesion. BCR-ABL also associates with the Janus kinase and signal transducer and activator of transcription (JAK–STAT) pathway. Finally, BCR-ABL activates pathways that lead to atypical responses to chemotactic factors, which leads to an increase in cell migration. BCR-ABL also associates with survival proteins that interact with the mitochondrial-based BCL2 family. CAS, p130 CRK-associated substrate; GAB2, GRB2-associated binding protein 2; SHIP, SH2-containing inositol-5-phosphatase

(Imatinib mesilato)

Copyright © 2011 Academic Press Inc. Figure 1.6 Copyright © 2011 Academic Press Inc.

Copyright © 2011 Academic Press Inc. Esempi di difese locali Figure 3.1 Copyright © 2011 Academic Press Inc.

Copyright © 2011 Academic Press Inc. Figure 1.2 Copyright © 2011 Academic Press Inc.

Copyright © 2011 Academic Press Inc. NECROSI DAMP: Damage associated molecular pattern Pattern recognition receptors Pathogen associated molecular pattern Figure 1.3 Copyright © 2011 Academic Press Inc.

Copyright © 2011 Academic Press Inc. Figure 1.4 Copyright © 2011 Academic Press Inc.

TRASDUZIONE DEL SEGNALE DA PARTE DI RECETTORI IMMUNI ITAM (Immunoreceptor tyrosine-based inhibitory motif) : DExxYxxL/I(x)6-7YxxL/I

FcgRIIA FcgRIIB ITIM: I/VxxYxxL/V ITAM Immunoreceptor tyrosine-based inhibitory motif

TRASDUZIONE DEL SEGNALE DA PARTE DI RECETTORI IMMUNI

attivazione

C-type lectins

Lyn/Hck/Fgr Syk Syk Lyn Syk Lyn Fyn/ Lck Syk ZAP70

Abl

Lipide fosfatasi

Protein fosfatasi

? ANCHE ALCUNE FAMIGLIE DI INTEGRINE (b2 E b3) TRASDUCONO IL SEGNALE MEDIANTE UN MODULO BASATO SU ADATTATORI CON SEQUENZA ITAM E SYK ? YP SYK SYK ITAM YP SFK